Extended-release drug delivery compositions

ABSTRACT

An extended-release drug delivery composition and method of administering the same is provided. The composition comprises microspheres loaded with a biologically-active agent and suspended in a soluble polymer capable of forming a film upon injection onto a biological surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/643,857 filed on Jul. 7, 2017, which is a continuation of U.S.application Ser. No. 14/738,174 filed on Jun. 12, 2015, which claimspriority to U.S. Provisional Application No. 62/011,380 filed on Jun.12, 2014, each of which is incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No.1R43DC012749-01 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

One of the challenges in drug delivery is the ability to target thetreatment to a particular area of the body or a particular biologicaltissue and maintain delivery at the area or tissue. In most instances,repetitive treatments are needed over the course of days to weeks inorder to maintain the necessary therapeutic level of an active agent.Simply stated, this approach is inconvenient and costly.

For example, sudden sensorineural hearing loss (SSNHL) is a disease thatattacks 4,000 Americans annually and is characterized by near completehearing loss in as little as a few hours. The most efficacious treatmentfor SSNHL consists of frequent injections of an anti-inflammatorysteroid into the middle ear, which diffuses into the inner ear via theround-window membrane (RWM). The invasive nature of these injections,especially if delivery is desired directly on the surface of the RWM,results in low patient compliance and a loss of efficacy. Thus, SSNHLpatients and patients having other conditions that require a localizedlong-term treatment protocol would benefit from a drug delivery platformthat permits the use of a single injection while providing anextended-release profile of the therapeutic agent.

SUMMARY

The present disclosure relates to an extended-release drug deliveryplatform. In one embodiment, an extended-release therapeutic compositionis provided. The composition comprises a film forming agent and abiologically active agent. The film forming agent can be a solublepolymer, whether in water or some other solvent, including, but notlimited to, ethanol, benzyl alcohol, or ethyl acetate. Examples ofsuitable polymers include, but are not limited to polyvinyl alcohol(PVA), polyvinyl acetate (PVAc), alginate, polyethylene glycol (PEG),hydroxypropyl methyl cellulose (HPMC), polyvinyl pyrrolidone (PVP),eudragits, collagen, and gelatin. The biologically active agent can be,for example, a therapeutic compound or a diagnostic agent. Thebiologically active agent can be loaded in a plurality of microspheresor microcapsules. The composition may further comprise a surfactant. Thesurfactant can be, for example, polysorbate or sorbitan laurate.

In another embodiment, a single-injection method for administration of acomposition to a target tissue is provided. The method comprises thestep of injecting a composition onto a biological tissue of a subject,wherein the composition comprises a film forming agent and abiologically active agent. In one instance, the biological tissue is theround window membrane of the inner ear and the step of injectingincludes the use of an intratympanic injection. The method may furthercomprise allowing the composition to form a film on the biologicaltissue by maintaining the subject in a position to facilitate retentionof the composition on the biological tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. Some specific example embodiments of thedisclosure may be understood by referring, in part, to the followingdescription and the accompanying drawings.

FIG. 1 describes (A) the process of intratympanic injection, whereas thefilm forming formulation is deposited directly on the RWM, (B)illustrates the attachment of the drug delivery system, in this instancewith microspheres, to the RWM, and (C) illustrates the replacement ofmultiple therapeutic injections with a single injection film formingdrug delivery system.

FIG. 2 shows (A) scanning electron micrographs of microspheres formedfrom emulsion methods, compared to (B,C,D) discrete sizes made byprecision particle fabrication technology, and (E) the relative sizedistribution of precision particle fabrication technology compared toemulsion methods whereas 90% of the PPF-produced microspheres (B-D) arewithin 2% of mean diameter (Scale bar=100 μm).

FIG. 3 shows betamethasone-loaded PLGA microspheres created withprecision particle fabrication technology with a size of approximately30 μm.

FIG. 4 shows that microspheres with film forming agent are adhered tothe RWM at (A) 21 and (C) 35 days post-injection, whereas microspheresinjected without a film forming component (i.e. only normal saline) arenot present on the RWM at (B) 21 or (D) 35 days post-injection.Scalebar=(A, B) 400 μm and (C, D)=300 μm.

FIG. 5 shows histological staining of the RWM surrounding tissue for theinflammatory protein tumor necrosis factor (TNF) alpha. Stainingintensity indicates that no significant inflammatory response waspresent in mice (A) with the film forming formulation compared to (B)without the film forming formulation.

FIG. 6 demonstrates release profiles for various active ingredients in acommon film forming agent without encapsulation of the activeingredient.

FIG. 7 demonstrates release profiles of dexamethasone from various filmforming agent formulations without encapsulation of the dexamethasone.

FIG. 8 demonstrates release profiles of various encapsulated activeingredients in a common film forming agent formulation.

FIG. 9 demonstrates release profiles of betamethasone encapsulated intwo different microsphere types in a common film forming agentformulation.

FIG. 10 demonstrates release profiles of betamethasone encapsulated inthree different sizes of microspheres in a common film forming agentformulation.

DESCRIPTION

The present disclosure provides an extended release drug deliveryplatform using a fast film forming agent (FFA). The FFA can be appliedto a surface of a target tissue where it is allowed to form a film. Thefilm retains a biologically-active agent that is then released over adesired period of time and in many instances, over weeks. This providesthe advantage of maintaining a proper localization for the treatment andalso permits a fine tuning of the release profile. In one embodiment,the FFA can be used to deliver drug-loaded microcapsules or microspheresto the round membrane window via an intratynpanic injection therebyeliminating the need for multiple, painful injections while providingfor more predictable drug levels within the inner ear perilymph fortreatment of inner ear disorders such as SSNHL. To this end, FIG. 1provides a representation of the intratympanic injection procedure inrelationship to the anatomy of the ear (panel A). Panel B of FIG. 1depicts intratympanic injection of drug-loaded microspheres (orangecircles) which localize to the round window membrane (RWM) using thepresent FFA composition (green squares). Using the present FFAcomposition, a delivery system for maintaining localization of atherapeutic agent to the RWM with a single injection is possible, incontrast to multiple injections. Thus, the present disclosure providescompositions comprising the FFA and methods of using the same to providean extended-release therapy for treatment of various disorders thatrequire long-term localized treatment, including inner ear disorders.

In one embodiment, a composition that provides an extended-releaseprofile is provided. The composition comprises a film forming agent anda biologically active agent.

The film-forming agent (FFA) is a means for forming a film on abiological tissue of a subject (also referred to herein as “film formingmeans” or “FFA means”). The film forming means of the present disclosureis capable of: (i) serving as the injectable carrier for abiologically-active agent, such as drug-loaded microspheres ormicrocapsules, and (ii) adhering the biologically-active agent to atarget membrane, biological tissue, or surface, such as the round windowmembrane (RWM) of the inner ear, for an extended period of time, forexample 20 to 35 days. The FFA means is comprised of Generally Acceptedas Safe (GRAS) materials and is readily soluble in water or otherappropriate solvents, allowing it to be delivered in a dry powdersyringe for resuspension immediately prior to use. The FFA means, in oneembodiment, comprise a soluble polymer such as polyvinyl alcohol (PVA),polyvinyl acetate (PVAc), alginate, polyethylene glycol (PEG),hydroxypropyl methyl cellulose (HPMC), polyvinyl pyrrolidone (PVP),eudragits, collagen, and gelatin. For example, in one embodiment, thefilm forming means is PVA having a molecular weight range of 20,000 to30,000 and in some instances may by hydrolyzed (e.g., 88% hydrolyzed).

In certain embodiments, the film forming means may comprise 0.5-10% w/vof the composition. However, it should be understood that the filmforming means may be formulated at different percentages based on theparticular polymer employed. For example, a FFA means comprising PEGcould account for 60-90% w/v of the composition.

The film forming means further comprises a carrier liquid. The carrierliquid utilized is dependent on the polymer or other substance used forthe film forming agent and may be a water or a solvent. Furthermore, thecarrier liquid should possess the ability to evaporate at physiologicaltemperatures, such as 37° C. Thus, the excess carrier liquid that doesnot form part of the film will evaporate quickly. The carrier liquid ofthe film forming means includes, for example, water, ethanol, benzylalcohol, and ethyl acetate.

The composition may further comprise a number of different excipientsincluding a surfactant, a stabilizer, a release modifier, or adensifier. For example, a surfactant is used in certain embodiments ofthe present composition based on the solubility of thebiologically-active agent. In the instance the biologically active agentis hydrophobic, polysorbate can be used as the surfactant. However,sorbitan laurate can be used in the instance the biologically-activeagent is hydrophilic. The surfactant can be polysorbate 20, polysorbate60, or polysorbate 80 based on the desired release profiles of thebiologically-active agent. The excipient may comprise approximately0.1%-10% w/v of the composition, and in some instance, 0.5-5% w/v of thecomposition.

Upon injection at the target surface, the film forming means generallyforms a film upon evaporation of the water or solvent content of thecomposition which, in some instances, will occur in about 20-50 minutespost-injection. It should be understood that use of the FFA means todeliver biologically-active agents to the inner ear is just oneembodiment of the technology and the FFA means could be used in avariety of other applications. Referring now to panel B of FIG. 1,examples of target biological surfaces or tissues in which the FFA meansis preferable are those surfaces, tissues, or membranes that provide abarrier between a predominately non-fluid or air-filled cavity and afluid-rich or tissue-dense region, such surfaces referred to herein asbiological barrier structures. Examples of biological barrier structuresare the round membrane window of the inner ear or the external surfaceof a nasal polyp. In these instances, the FFA means is applied to theside of the membrane, tissue, or surface exposed to the non-fluid orair-filled cavity which permits the FFA means to dry and form a film onthe surface. The liquid or tissue on the other side of the membrane,surface, or tissue facilitates diffusion of the biologically-activeagent from the FFA means into the fluid-filled area thereby targetingthe therapy or diagnostic to the target region.

The biologically-active agent may comprise a therapeutic or diagnosticcompound and thus may be, for example, a steroid, antibody, peptide,nucleic acid, antioxidant, chemical, small molecule, and other similarcompounds. In one embodiment, the biologically-active agent isbetamethasone, dexamethasone, or penicilin.

The biologically-active agent can be formulated as nanoparticle ormicrosphere. For example, the biologically active compound may befabricated using the Precision Particle Formation (PPF) method asdescribed in U.S. Pat. Nos. 6,669,961, 7,368,130, and 7,309,500, all ofwhich are incorporated by reference herein in their entireties. Briefly,in this method, a drug-matrix solution is sprayed through a nozzle with(i) vibrational excitation to produce uniform droplets, and (ii) anannular, non-solvent carrier stream to reduce the diameter of theexiting jet. The microspheres can be formed of poly(D,L-lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA),poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(ethyleneglycol) (PEG), poly(vinyl acetate) (PVAc), ethyl cellulose, or similarbiodegradable polymers compatible with precision particle fabricationtechnology as described in U.S. Pat. Nos. 6,669,961, 7,368,130, and7,309,500.

Alternatively, in certain other embodiments, rather than comprising ahomogenous mixture of the biologically-active agent and polymermaterials, the microsphere may comprise a hydrophobic matrix layer and acore, wherein the biologically-active agent is dispersed within the coreand is surround by the hydrophobic matrix layer. These microspheres mayalso be fabricated using the PPF method referred to above. Thehydrophobic matrix may be a hydrophobic wax material, a lipid material,a glycol polymer, or a combination thereof. In certain embodiments,suitable hydrophobic matrix materials have a melting point at or aboveabout 45° C. and a viscosity when melted sufficient to allow spraying.

Suitable lipid materials should be solid at room temperature and have amelting temperature at or above about 45° C. Examples of suitable lipidmaterials include, but are not limited to, glycerol fatty acid esters,such as triacylglycerols (e.g., tripalmitin, tristearin, glyceryltrilaurate, coconut oil), hydrogenated fats, ceramides, and organicesters from and/or derived from plants, animals, minerals.

Suitable glycol polymers should be solid at room temperature and have amelting temperature at or above about 45° C. Examples of suitable glycolpolymers include, but are not limited to, high molecular weight glycols(e.g., polyethylene glycol with a minimum of 20 repeating units),cellulose ethers (e.g., ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, microcrystalline cellulose), celluloseesters (e.g., cellulose acetate, cellulose acetate phthalate,hydroxypropyl methyl cellulose phthalate), polyacrylates derivatives,polymethacrylates derivatives, poloxamers, and starch and itsderivatives.

In certain embodiments, the hydrophobic matrix may be a hydrophobic waxmaterial. The hydrophobic wax matrix may be any wax-like materialsuitable for use with the active ingredient. Examples of suitablehydrophobic waxes include, but are not limited to, ceresine wax,beeswax, ozokerite, microcrystalline wax, candelilla wax, montan wax,carnauba wax, paraffin wax, cauassu wax, Japan wax, and Shellac wax.

In certain embodiments of the present composition employing ahydrophobic wax matrix, the microspheres further comprise a densifier. Adensifier may used to increase the density of a particle. For example, adensifier may be used to make a particle heavier so that it willapproach or be closer to the density of a liquid vehicle in which themicrospheres may be suspended. Examples of suitable densifiers include,but are not limited to, titanium dioxide, calcium phosphate, and calciumcarbonate. In one embodiment, the one or more densifiers may be presentin the microspheres in an amount in the range of from about 0% to about40% by weight of the microspheres.

The hydrophobic matrix may be present in the microspheres in an amountin the range of from about 5% to about 90%, about 5% to about 30%, about20% to about 80%, or about 40% to about 60% by weight of themicrocapsule. In another embodiment, the hydrophobic matrix may bepresent in the microcapsule in an amount sufficient to provide sustainedrelease of the active ingredient over a period ranging between about 1hour to about 12 hours or more. For example, the wax may be present inthe microspheres in an amount sufficient to provide sustained release ofthe hydrophilic active ingredient over a period of about 1 hour, 2hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24hours, or longer. In certain embodiments, the hydrophobic matrix may beincreased or decreased depending on the particular releasecharacteristics desired. In addition, more than one hydrophobic matrixlayer may be used to achieve the particular sustained release desired.In general, higher hydrophobic matrix concentrations favor longer, moresustained release of the active ingredient and lower concentrationsfavor faster, more immediate release.

In certain embodiments, the microspheres of the present disclosurecomprise a stabilizer. The stabilizer may improve the properties of thehydrophobic wax matrix and provide improved stability of themicrospheres over time, as well as improved dissolution profiles.Changes in microspheres can occur over time that affect the particle'sperformance. Such changes include physical, chemical, or dissolutioninstability. These changes are undesirable as they can affect aformulation's shelf stability, dissolution profile, and bioavailabilityof the active ingredient. For example the hydrophobic wax matrix oractive ingredient may relax into a lower energy state, the particle maybecome more porous, and the size and interconnectivity of pores maychange. Changes in either the active ingredient or hydrophobic waxmatrix may affect the performance of the particle. The presentdisclosure is based, at least in part, on the observation that astabilizer added to the hydrophobic wax matrix improves the stabilityand performance of the microspheres of the present disclosure. By way ofexplanation, and not of limitation, it is believed that the stabilizerinteracts with the hydrophobic wax material making it resistant tophysical changes. Accordingly, the microspheres of the presentdisclosure comprise a stabilizer. Examples of suitable stabilizersinclude but are not limited to, cellulose, ethyl cellulose,hydroxyproylmethyl cellulose, microcrystalline cellulose, celluloseacetate, cellulose phthalate, methyl cellylose, chitin, chitosan,pectin, polyacrylates, polymethacrylates, polyvinyl acetate, Elvax® EVAresins, acetate phthalate, polyanhydrides. polyvinylalcohols, siliconeelastomers, and mixtures thereof. Stabilizers may be used alone or incombination. The stabilizer may be present in the microspheres in anamount from about 0.1% to about 10% by weight of the particle. Forexample, the stabilizer may be present in an amount from about 0.1% toabout 5%, about 0.5% to about 2.5%, and about 5% to about 10% by weightof the particle.

In certain embodiments, the microspheres of the present disclosure alsocomprise a release modifier. The present disclosure is also based on theobservation that a release modifier improves the performance ofhydrophobic wax matrix microspheres particularly during the later stagesof the active ingredient's release. The release modifier is believedalso to interact with the stabilizer (e.g., improve the stabilizer'ssolubility) to facilitate preparation of the microspheres. It is alsobelieved that the release modifier may adjust the relativehydrophobicity of the hydrophobic wax material. Examples of suitablerelease modifiers include but are not limited to, stearic acid, sodiumstearate, magnesium stearate, glyceryl monostearate, cremophor (castoroil), oleic acid, sodium oleate, lauric acid, sodium laurate, myristicacid, sodium myristate, vegetable oils, coconut oil, mono-, di-,tri-glycerides, stearyl alcohol, span 20, span 80, and polyethyleneglycol (PEG). Release modifiers may be used alone or in combination. Forexample, in certain embodiments, the release modifier may be acombination of stearic acid and glyceryl mono stearate. The releasemodifier may be present in the microspheres in an amount from about 0.5%to about 90% by weight of the particle. For example, the releasemodifier may be present in an amount from about 0.5% to about 10%, about1% to about 5%, about 2.5% to about 5%, about 5% to about 10%, about 10%to about 25%, about 20% to about 90%, about 40% to about 80%, about 50%to about 70%, about 60% to about 80%, and about 80% to about 90% byweight of the particle. In general, higher release modifierconcentrations favor faster release of the active ingredient and lowerconcentrations favor longer, sustained release.

Moreover, in certain embodiments, the microspheres used in the presentcompositions can have a particle size diameter of less than 150 μm, lessthan 100 μm, less than 50 μm, and more preferably for use inintratympanic injections, the microspheres can have a particle sizediameter of about 30 μm to about 60 μm. Thus, the compositions of thepresent invention, in certain embodiments, comprise a plurality ofmicrospheres having a mean particle diameter of less than 150 μm, lessthan 100 μm, less than 50 μm, and more preferably for use inintratympanic injections, the microspheres can have a particle sizediameter of about 30 μm to about 60 μm.

The size and size uniformity of drug-encapsulated microspheres directlycontrols their release profiles. Very small particles (<5 μm or so),often called “fines”, release drug quickly, leading to an initial“burst” release effect. Very large particles (>500 μm), on the otherhand, tend to slowly release drug over a prolonged period of time.Polydisperse mixtures of drug-encapsulated particles, therefore, offerpoor control of drug release. The Precision Particle Fabricationtechnology produces uniform microspheres that allow for precise controlof release properties. Thus, the microspheres of the present compositionmay possess a particle size distribution that deviates from the meanparticle diameter by 10% or less, by 5% or less, or by 2% or less asshown in FIG. 2 (panels B, C, D, and E). Comparatively, otherencapsulation methods, including precipitation, phase separation, and/oremulsion technique (panel A) demonstrate standard deviations equal to25-50% or more of the mean as shown in panel E of FIG. 2.

One exemplary composition of the present disclosure is provided fordelivering at least 1.0 μg of a biologically-active compound to the RWMover 30 days. Here, the FFA is 10% w/v Poly(vinyl alcohol)(Mw:20,000-30,000, 88% hydrolyzed), the surfactant is 5.0% w/v Tween 80, andthe biolgocially-active agent is betamethasone. The betamethasone isloaded in microspheres (approximately 30 μm particle size) using the PPFmethod at any one of the following loading and particle concnetrationsin a 2 μL total composition volume: (1) 10 mg/ml particle concentrationwith a particle loading of 5.0% betamethasone; (2) 50 mg/ml particleconcentration with a particle loading of 1.0% betamethasone; and (3) 100mg/ml particle concentration with a particle loading of 0.5%betamethasone. Other specific formulations of the present compositionsare presented in the examples below.

Thus, in one embodiment, an injectable composition is provided. Theinjectable composition of the present embodiment comprises a means forforming a film on a biological tissue. The means comprises a solublepolymer and a carrier liquid, wherein the soluble polymer is from 0.5%to about 10% w/v of the composition, and wherein the carrier liquid isable to evaporate at 37° C. The composition further comprises abiologically-active agent. The composition may further comprise anexcipient, wherein the excipient is from about 0.5 to about 5% w/v ofthe composition.

In certain embodiments, the soluble polymer is selected from the groupconsisting of polyvinyl alcohol (PVA), polyvinyl acetate (PVAc),alginate, polyethylene glycol (PEG), hydroxypropyl methyl cellulose(HPMC), polyvinyl pyrrolidone (PVP), eudragits, collagen, and gelatin.

In certain embodiments, the carrier liquid is selected from the groupconsisting of water, ethanol, benzyl alcohol, and ethyl acetate.

In certain embodiments, the excipient is a surfactant selected from thegroup consisting of polysorbate 20, polysorbate 60, polysorbate 80 orsorbitan laurate.

In certain embodiments, the biologically-active agent is selected fromthe group consisting of a steroid, an antibody, a peptide, a nucleicacid, an antioxidant, and a small molecule. In certain embodiments, thebiologically-active agent is betamethasone or dexamethasone.

In certain embodiments, the injectable composition further comprises amicrosphere, wherein the biologically-active agent is present in themicrosphere. The microsphere further comprises a material selected fromselected from the group consisting of poly (D,L-lactic-co-glycolic acid)(PLGA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA),poly(caprolactone) (PCL), poly(ethylene glycol) (PEG), poly(vinylacetate) (PVAc), and ethyl cellulose.

In certain embodiments, the microspheres of the present compositionpossess a mean particle diameter of less than 150 μm, less than 100 μm,less than 50 μm, and in some instances, from about 30 μm to about 60 μm.In certain embodiments, 90% of the microspheres of the compositioncomprise a particle diameter that does not deviate from the meanparticle diameter by more than 10%, 5%, or 2%.

A single-injection method for administration of a composition of thepresent disclosure to a biological barrier structure is also provided.The method can be performed using any of the above-describedcompositions. The composition can be loaded in a syringe or catheter.Due to the small particle sizes (30 μm) that can be generated using thePPF technology, syringe gauges of 28 and 30 can be used. In oneembodiment, a method for administration of an extended-releasetherapeutic is provided. The method comprises injecting one of thecompositions described herein onto a biological barrier structure andallowing the composition to form a film on the biological barrierstructure. In a specific embodiment, a method of treating disorders ofthe inner ear is provided. In this embodiment, intratympanic injectionis accomplished with standard out-patient steroid solutionadministration techniques, requiring minimal extra effort on the part ofphysicians and patients. The composition is injected onto the surface ofthe RWM where it forms a film following evaporation of the water orother solvent used to carry the composition. The film allows thetherapeutic, for example, betamethasone, to be retained at the RWM forextended periods of time thereby providing a single injection method forlong-term administration of the therapeutic to the inner ear.

In certain instance, the method may further comprise the step ofmaintaining the subject in a position during the injection and for atime period following the injection sufficient to permit the compositionto form a film on the round window membrane. In certain embodiments, thesubject is suffering from SSNHL. In instances where the method isperformed on a subject suffering from SSNHL, examples ofbiologically-active agents include, but are not limited to steroids suchas betamethasone or dexamethasone, antioxidants such as vitamin A,vitamin C, and vitamin E, and nucleic acids such as siRNA directed toreduce expression of genes that restrict hair cell proliferation andother gene targets that otherwise would prevent regeneration of haircells as well as gene therapies that would promote hair cellregeneration.

EXAMPLES

The examples herein are illustrations of various embodiments of thepresent invention and are not intended to limit it in any way.

Example 1: In Vivo Analysis of FFA: Localization and InflammatoryResponse

Uniform betamethasone-loaded biodegradable microspheres were preparedusing the Precision Particle Fabrication technology. Briefly,betamethasone was dissolved in dichloromethane (DCM), to which a 50:50poly (D,L-lactic-co-glycolic acid) (PLGA) was added such that thebetamethasone comprised 1.0% w/w of the total solids content. Thesuspension was loaded into a syringe pump and used to producemicrospheres using the Precision Particle Fabrication (PPF) nozzle. Themicrospheres were collected in a solution of poly (vinyl alcohol) indeionized (DI) water. Following a 3-hour solvent evaporation step, theparticles were filtered and lyophilized for 48 hours. The resultingparticles are shown in FIG. 3.

Betamethasone-loaded microspheres suspended in the FFA were delivered tomice RWM as follows. C57/BL6 mice were anesthetized with aKetamine/Xylazine cocktail, laid on their side, and immobilized. Theskin and soft tissue was retracted, and an access hole to the tympaniccavity was created with a 28 GA needle. ˜2.0 μL injections of 50 mg/mLfluorescent dye-loaded microspheres were then delivered directly abovethe RWM with a 10 μL Hamilton syringe. The mice were kept in thisposition for 5 minutes before being sutured and imaged on an IVIS invivo Imaging System (Perkin-Elmer, Waltham Mass.) to confirmlocalization of the formulation to the inner ear space, and brought outof anesthesia. Negative control mice were injected with fluorescentmicrospheres without the FFA component (saline vehicle). At 21 and 35days timepoints, mice were sacrificed and necropsy was performed toevaluate microsphere localization.

To measure potential inflammatory response, mice were euthanized at 28days, and the inner ear anatomy was isolated, removed, decalcified, andparaffin-embedded. Samples were sectioned and immunohistochemicallystained for two major inflammatory markers, interleukin (IL)-6 and tumornecrosis factor (TNF)-α, in addition to hematoxylin and eosin (H&E). Invitro dissolution testing of betamethasone-loaded microspheresdemonstrated that microsphere size plays a role in controlling therelease of drug from the microspheres.

Referring now to FIG. 4, mice treated with microspheres suspended in theFFA had microspheres localized directly on the RWM at 21 days with athin film as intended (panel A). There appeared to be only a slight lossof sphericity, indicating that some degradation of the particlesoccurred, but the overall integrity of the delivery system wasmaintained. Negative control mice displayed no visible microspheres,indicating that the particles had migrated away from the surgical sitedue to lack of an FFA component (panel B). Similarly, at 35 days, ananalogous set of mice were euthanized and dissected. Once again, we wereable to see a deposition of microspheres in the space directly adjacentto the RWM in mice treated with microspheres suspended in the FFA (panelC), and an absence of microspheres in negative control mice (panel D).

Staining indicated that the microspheres and FFA caused no significantinflammatory response. The intensity of TNF-α and IL-6 development wassimilar between the groups (data not shown), and H&E revealed nodiscernible changes in hair cell anatomy or apparent tissue reaction asshown in FIG. 5, panels A and B.

Suspending betamethasone-loaded microspheres a film forming agentprovided an injectable, extended release intratympanic delivery systemthat can localize drug to the RWM of mice for greater than 35-days withminimal inflammatory response.

Example 2: Various Pharmaceutical Ingredient Release from OneFilm-Forming Agent (FFA) Type

This example illustrates how one film-forming agent type can accommodaterelease of many pharmaceutical ingredients, without the need for amicrosphere component. They are displayed in FIG. 6.

Dexamethasone

An FFA was made, consisting of 10% w/v poly(vinyl alcohol) and 5% v/vTween 80 dissolved in deionized water. Dexamethasone was dispersedwithin the film forming agent via sonication at a concentration of 1mg/mL. On a Franz cell apparatus fitted with a cellulose acetatemembrane and 5% w/v Brij O20 receptor phase, 1.0 mL of thedexamethasone/FFA was deposited. Drug diffusion across the membrane at37° C. was measured for 14 days, and quantified with HPLC. The drugrelease was normalized to total detected dexamethasone at 14 days.

Penicillin

An FFA was made, consisting of 10% w/v poly(vinyl alcohol) and 5% v/vTween 80 dissolved in deionized water. Penicillin was dispersed withinthe film forming agent via sonication at a concentration of 1 mg/mL. Ona Franz cell apparatus fitted with a cellulose acetate membrane and 5%w/v Brij O20 receptor phase, 1.0 mL of the penicillin/FFA was deposited.Drug diffusion across the membrane at 37° C. was measured for 14 days,and quantified with HPLC. The drug release was normalized to totaldetected penicillin at 14 days.

siRNA

An FFA was made, consisting of 10% w/v poly(vinyl alcohol) and 5% v/vTween 80 dissolved in deionized water. SiRNA was dispersed within thefilm forming agent via sonication at a concentration of 1 μg/mL. On aFranz cell apparatus fitted with a cellulose acetate membrane and 5% w/vBrij O20 receptor phase, 1.0 mL of the SiRNA/FFA was deposited. Drugdiffusion across the membrane at 37° C. was measured for 14 days, andquantified with a picogreen assay. The drug release was normalized tototal detected SiRNA at 14 days.

DNA

An FFA was made, consisting of 10% w/v poly(vinyl alcohol) and 5% v/vTween 80 dissolved in deionized water. DNA was dispersed within the filmforming agent via sonication at a concentration of 1 μg/mL. On a Franzcell apparatus fitted with a cellulose acetate membrane and 5% w/v BrijO20 receptor phase, 1.0 mL of the DNA/FFA was deposited. Drug diffusionacross the membrane at 37° C. was measured for 14 days, and quantifiedwith a picogreen assay. The drug release was normalized to totaldetected DNA at 14 days.

Example 3: One Pharmaceutical Ingredient Release from MultipleFilm-Forming Agent (FFA) Types

This example illustrates how multiple film-forming agent types canaccommodate release of a single pharmaceutical ingredient, without theneed for a microsphere component. They are displayed in FIG. 7.

10% PVA+5% Tween 80

A FFA was made, consisting of 10% w/v poly(vinyl alcohol) and 5% v/vTween 80 dissolved in deionized water. Dexamethasone was dispersedwithin the film forming agent via sonication at a concentration of 1mg/mL. On a Franz cell apparatus fitted with a cellulose acetatemembrane and 5% w/v Brij O20 receptor phase, 1.0 mL of thedexamethasone/FFA was deposited. Drug diffusion across the membrane at37° C. was measured for 5 days, and quantified with HPLC. The drugrelease was normalized to total detected dexamethasone at 5 days.

60% PEG 2000 in H2O

A FFA was made, consisting of 60% w/v poly(ethylene glycol) 2000dissolved in deionized water. Dexamethasone was dispersed within thefilm forming agent via sonication at a concentration of 1 mg/mL. On aFranz cell apparatus fitted with a cellulose acetate membrane and 5% w/vBrij O20 receptor phase, 1.0 mL of the dexamethasone/FFA was deposited.Drug diffusion across the membrane at 37° C. was measured for 5 days,and quantified with HPLC. The drug release was normalized to totaldetected dexamethasone at 5 days.

2% Ethylcellulose in Ethyl Acetate

A FFA was made, consisting of 2% ethylcellulose dissolved in ethylacetate. Dexamethasone was dispersed within the film forming agent viasonication at a concentration of 1 mg/mL. On a Franz cell apparatusfitted with a cellulose acetate membrane and 5% w/v Brij O20 receptorphase, 1.0 mL of the dexamethasone/FFA was deposited. Drug diffusionacross the membrane at 37° C. was measured for 5 days, and quantifiedwith HPLC. The drug release was normalized to total detecteddexamethasone at 5 days.

0.67% HPMC in Ethanol

A FFA was made, consisting of 0.67% hydroxyproplymethylcellulose (HPMC)dissolved in ethanol. Dexamethasone was dispersed within the filmforming agent via sonication at a concentration of 1 mg/mL. On a Franzcell apparatus fitted with a cellulose acetate membrane and 5% w/v BrijO20 receptor phase, 1.0 mL of the dexamethasone/FFA was deposited. Drugdiffusion across the membrane at 37° C. was measured for 5 days, andquantified with HPLC. The drug release was normalized to total detecteddexamethasone at 5 days.

2% PVAc in Benzyl Alcohol

A FFA was made, consisting of 2% poly(vinyl acetate) dissolved in benzylalcohol. Dexamethasone was dispersed within the film forming agent viasonication at a concentration of 1 mg/mL. On a Franz cell apparatusfitted with a cellulose acetate membrane and 5% w/v Brij O20 receptorphase, 1.0 mL of the dexamethasone/FFA was deposited. Drug diffusionacross the membrane at 37° C. was measured for 5 days, and quantifiedwith HPLC. The drug release was normalized to total detecteddexamethasone at 5 days.

Example 4: Various Pharmaceutical Ingredient Release from OneMicrosphere Type

The following examples illustrate how one microsphere type canaccommodate release of a multiple pharmaceutical ingredients, withoutthe need for a film forming agent component. They are displayed in FIG.8.

Dexamethasone

Dexamethasone was dispersed in a polymer solution consisting of 5050PLGA (I.V. 0.45 dL/g with ester end group) dissolved in dichloromethane.The drug/polymer solution was processed with precision particlefabrication to make microspheres of ˜40 μm, which were collected,filtered, and lyophilized. On a Franz cell apparatus fitted with acellulose acetate membrane and 5% w/v Brij O20 receptor phase, ˜20 mg ofthe dexamethasone-loaded microspheres were deposited. Drug diffusionacross the membrane at 37° C. was measured for 35 days, and quantifiedwith HPLC. Microspheres released approximately 1.7 μg dexamethasone, andthe drug release was normalized to total detected dexamethasone at 35days.

Penicillin

Penicillin was dispersed in a polymer solution consisting of 5050 PLGA(I.V. 0.45 dL/g with ester end group) dissolved in dichloromethane. Thedrug/polymer solution was processed with precision particle fabricationto make microspheres of ˜40 μm, which were collected, filtered, andlyophilized. On a Franz cell apparatus fitted with a cellulose acetatemembrane and 5% w/v Brij O20 receptor phase, ˜20 mg of thepenicillin-loaded microspheres were deposited. Drug diffusion across themembrane at 37° C. was measured for 35 days, and quantified with HPLC.Microspheres released approximately 77 μg penicillin, and the drugrelease was normalized to total detected penicillin at 35 days.

Albumin

Albumin was dissolved in water, then dispersed by sonication into apolymer solution consisting of 5050 PLGA (I.V. 0.45 dL/g with ester endgroup) dissolved in dichloromethane. The protein/polymer solution wasprocessed with precision particle fabrication to make microspheres of˜40 μm, which were collected, filtered, and lyophilized. On a Franz cellapparatus fitted with a cellulose acetate membrane and 5% w/v Brij O20receptor phase, ˜20 mg of the albumin-loaded microspheres weredeposited. Protein diffusion across the membrane at 37° C. was measuredfor 35 days, and quantified with a micro-BCA assay. Microspheresreleased approximately 4.9 mg albumin, and the protein release wasnormalized to total detected albumin at 35 days.

SiRNA

SiRNA was dissolved in water, then dispersed by sonication into apolymer solution consisting of 5050 PLGA (I.V. 0.45 dL/g with ester endgroup) dissolved in dichloromethane. The nucleic acid/polymer solutionwas processed with precision particle fabrication to make microspheresof ˜40 μm, which were collected, filtered, and lyophilized. On a Franzcell apparatus fitted with a cellulose acetate membrane and 5% w/v BrijO20 receptor phase, ˜20 mg of the SiRNA-loaded microspheres weredeposited. Nucleic acid diffusion across the membrane at 37° C. wasmeasured for 35 days, and quantified with a picogreen assay.Microspheres released approximately 1.1 μg, and the nucleic acid releasewas normalized to total detected SiRNA at 35 days.

DNA

DNA was dissolved in water, then dispersed by sonication into a polymersolution consisting of 5050 PLGA (I.V. 0.45 dL/g with ester end group)dissolved in dichloromethane. The nucleic/polymer solution was processedwith precision particle fabrication to make microspheres of ˜40 μm,which were collected, filtered, and lyophilized. On a Franz cellapparatus fitted with a cellulose acetate membrane and 5% w/v Brij O20receptor phase, ˜20 mg of the DNA-loaded microspheres were deposited.Nucleic acid diffusion across the membrane at 37° C. was measured for 35days, and quantified with a picogreen assay. Microspheres releasedapproximately 1.4 μg, and the nucleic acid release was normalized tototal detected DNA at 35 days.

Example 5: One Pharmaceutical Ingredient Release from DifferentMicrosphere Types in a Single Film-Forming Agent Type

This example illustrates how different microsphere types can changerelease of a single pharmaceutical ingredient, without the need forchanging the film forming agent component. They are displayed in FIG. 9.

5050 PLGA 3A

Betamethasone was dispersed in a polymer solution consisting of 5050PLGA (I.V. 0.30 dL/g with acid end group) dissolved in dichloromethane.The drug/polymer solution was processed with precision particlefabrication and the microspheres were collected, filtered, andlyophilized. On a Franz cell apparatus fitted with a cellulose acetatemembrane and 5% w/v Brij O20 receptor phase, ˜10 mg of thebetamethasone-loaded microspheres were suspended in 100 uL of 10% w/vpoly(vinyl alcohol) and 5% v/v Tween 80 film-forming agent anddeposited. Drug diffusion across the membrane at 37° C. was measured for8 days, and quantified with HPLC. The drug release was normalized tototal entrapped drug, which was approximately 0.75% w/w of themicrospheres.

5050 PLGA 4.5E

Betamethasone was dispersed in a polymer solution consisting of 5050PLGA (I.V. 0.45 dL/g with ester end group) dissolved in dichloromethane.The drug/polymer solution was processed with precision particlefabrication and the microspheres were collected, filtered, andlyophilized. On a Franz cell apparatus fitted with a cellulose acetatemembrane and 5% w/v Brij O20 receptor phase, ˜10 mg of thebetamethasone-loaded microspheres were suspended in 100 uL of 10% w/vpoly(vinyl alcohol) and 5% v/v Tween 80 film-forming agent anddeposited. Drug diffusion across the membrane at 37° C. was measured for8 days, and quantified with HPLC. The drug release was normalized tototal entrapped drug, which was approximately 0.38% w/w of themicrospheres.

Example 6: One Pharmaceutical Ingredient Release from DifferentMicrosphere Sizes in a Single Film-Forming Agent Type

This example illustrates how different microsphere sizes can changerelease of a single pharmaceutical ingredient, without the need forchanging the film forming agent component or microsphere materialchemistry. They are displayed in FIG. 10.

40 μm

Betamethasone was dispersed in a polymer solution consisting of 5050PLGA (I.V. 0.30 dL/g with acid end group) dissolved in dichloromethane.The drug/polymer solution was processed with precision particlefabrication to make microspheres of ˜40 μm, which were collected,filtered, and lyophilized. On a Franz cell apparatus fitted with acellulose acetate membrane and 5% w/v Brij O20 receptor phase, ˜10 mg ofthe betamethasone-loaded microspheres were suspended in 100 uL of 10%w/v poly(vinyl alcohol) and 5% v/v Tween 80 film-forming agent anddeposited. Drug diffusion across the membrane at 37° C. was measured for28 days, and quantified with HPLC. Microspheres contained approximately0.38% w/w betamethasone, and the drug release was normalized to totaldetected betamethasone at 28 days.

50 μm

Betamethasone was dispersed in a polymer solution consisting of 5050PLGA (I.V. 0.30 dL/g with acid end group) dissolved in dichloromethane.The drug/polymer solution was processed with precision particlefabrication to make microspheres of ˜50 μm, which were collected,filtered, and lyophilized. On a Franz cell apparatus fitted with acellulose acetate membrane and 5% w/v Brij O20 receptor phase, ˜10 mg ofthe betamethasone-loaded microspheres were suspended in 100 uL of 10%w/v poly(vinyl alcohol) and 5% v/v Tween 80 film-forming agent anddeposited. Drug diffusion across the membrane at 37° C. was measured for28 days, and quantified with HPLC. Microspheres contained approximately0.65% w/w betamethasone, and the drug release was normalized to totaldetected betamethasone at 28 days.

60 μm

Betamethasone was dispersed in a polymer solution consisting of 5050PLGA (I.V. 0.30 dL/g with acid end group) dissolved in dichloromethane.The drug/polymer solution was processed with precision particlefabrication to make microspheres of ˜60 μm, which were collected,filtered, and lyophilized. On a Franz cell apparatus fitted with acellulose acetate membrane and 5% w/v Brij O20 receptor phase, ˜10 mg ofthe betamethasone-loaded microspheres were suspended in 100 uL of 10%w/v poly(vinyl alcohol) and 5% v/v Tween 80 film-forming agent anddeposited. Drug diffusion across the membrane at 37° C. was measured for28 days, and quantified with HPLC. Microspheres contained approximately0.69% w/w betamethasone, and the drug release was normalized to totaldetected betamethasone at 28 days.

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Theparticular embodiments disclosed above are illustrative only, as thepresent invention may be modified and practiced in different butequivalent manners apparent to those skilled in the art having thebenefit of the teachings herein. Furthermore, no limitations areintended to the details of construction or design herein shown, otherthan as described in the claims below. It is therefore evident that theparticular illustrative embodiments disclosed above may be altered ormodified and all such variations are considered within the scope andspirit of the present invention.

While compositions and methods are described in terms of “comprising,”“containing,” or “including” various components or steps, thecompositions and methods can also “consist essentially of” or “consistof” the various components and steps.

All numbers and ranges disclosed above may vary by some amount. Whenevera numerical range with a lower limit and an upper limit is disclosed,any number and any included range falling within the range isspecifically disclosed. In particular, every range of values (of theform, “from about a to about b,” or, equivalently, “from approximately ato b,” or, equivalently, “from approximately a-b”) disclosed herein isto be understood to set forth every number and range encompassed withinthe broader range of values.

Also, the terms in the claims have their plain, ordinary meaning unlessotherwise explicitly and clearly defined by the patentee. Moreover, theindefinite articles “a” or “an”, as used in the claims, are definedherein to mean one or more than one of the element that it introduces.If there is any conflict in the usages of a word or term in thisspecification and one or more patent or other documents that may beincorporated herein by reference, the definitions that are consistentwith this specification should be adopted.

What is claimed is:
 1. A method for treating a subject suffering from adisorder of the inner ear, comprising: injecting a composition to abiological tissue of the ear of the subject no more than one time in a14 day period, wherein the composition comprises a biologically-activeagent dispersed in a film-forming agent, wherein the film-forming agentcomprises a soluble polymer and a carrier liquid, wherein thefilm-forming agent is free of solvent, wherein the carrier liquid iscapable of evaporating at a temperature of about 37° C., and wherein thefilm-forming agent is capable of forming a film on the biological tissueupon evaporation of at least a portion of the carrier liquid.
 2. Themethod of claim 1, further comprising, after injecting the compositionto the biological tissue: allowing the at least a portion of the carrierliquid to evaporate, whereby the film-forming agent forms a film on thebiological tissue.
 3. The method of claim 1, wherein the soluble polymeris selected from the group consisting of polyvinyl alcohol (PVA),polyvinyl acetate (PVAc), alginate, polyethylene glycol (PEG),hydroxypropyl methyl cellulose (HPMC), polyvinyl pyrrolidone (PVP), aneudragit, collagen, gelatin and combinations thereof.
 4. The method ofclaim 1, wherein the carrier liquid is selected from the groupconsisting of water, ethanol, benzyl alcohol, and ethyl acetate.
 5. Themethod of claim 1, wherein the soluble polymer is from about 0.5%-10%w/v of the film-forming agent.
 6. The method of claim 1, wherein thebiological tissue is selected from the middle ear or the round windowmembrane of the inner ear.
 7. A method for treating a subject sufferingfrom a disorder of the inner ear, comprising: injecting a composition toa biological tissue of the ear of the subject no more than one time in a14 day period, wherein the composition comprises a biologically-activeagent dispersed in a film-forming agent, wherein the film-forming agentcomprises polyvinyl alcohol and a carrier liquid, wherein the polyvinylalcohol has a molecular weight of from about 20,000 to 30,000 Daltons,wherein the film-forming agent is free of solvent, wherein the carrierliquid is capable of evaporating at a temperature of about 37° C.,wherein the film-forming agent is capable of adhering to the biologicaltissue for an extended period of time, and wherein said extended periodof time is at least 14 days.
 8. The method of claim 7, furthercomprising, after injecting the composition to the biological tissue:allowing the at least a portion of the carrier liquid to evaporate,whereby the film-forming agent forms a film on the biological tissue. 9.The method of claim 7, wherein the carrier liquid is selected from thegroup consisting of water, ethanol, benzyl alcohol, and ethyl acetate.10. The method of claim 7, wherein the PVA is from about 0.5%-10% w/v ofthe film-forming agent.
 11. The method of claim 7, wherein thebiological tissue is selected from the middle ear or the round windowmembrane of the inner ear.
 12. A method for treating a subject sufferingfrom sensorineural hearing loss, comprising: injecting a composition toa biological tissue of the ear of the subject no more than one time in a14 day period, wherein the composition comprises a biologically-activeagent dispersed in a film-forming means.
 13. The method of claim 12,wherein the biological tissue is selected from the middle ear or theround window membrane of the inner ear.